Control of the use of performance-affecting substances in the horse is critical to the integrity of a wide range of equine sports, with major implications for both animal welfare and revenue streams. One class of medications enjoying particular public notoriety is the anabolic-androgenic steroid group, as highlighted by the recent 'Big Brown' affair and Congressional inquiries into the use of steroids in professional sports, including horse racing, in the USA. This review examines the latest developments pertaining to the analytical detection of these substances in equine biological samples and the supporting regulatory environment. Consideration is given to the full variety of sample matrices available, together with modern sample preparative approaches and instrumental techniques. Issues concerning the regulation of endogenous steroids, including thresholds where applicable, are also discussed.